Rho GTPase signalling networks in cancer cell transendothelial migration

Abstract

Rho GTPases are small signalling G-proteins that are central regulators of cytoskeleton dynamics, and thereby regulate many cellular processes, including the shape, adhesion and migration of cells. As such, Rho GTPases are also essential for the invasive behaviour of cancer cells, and thus involved in several steps of the metastatic cascade, including the extravasation of cancer cells. Extravasation, the process by which cancer cells leave the circulation by transmigrating through the endothelium that lines capillary walls, is an essential step for metastasis towards distant organs. During extravasation, Rho GTPase signalling networks not only regulate the transmigration of cancer cells but also regulate the interactions between cancer and endothelial cells and are involved in the disruption of the endothelial barrier function, ultimately allowing cancer cells to extravasate into the underlying tissue and potentially form metastases. Thus, targeting Rho GTPase signalling networks in cancer may be an effective approach to inhibit extravasation and metastasis. In this review, the complex process of cancer cell extravasation will be discussed in detail. Additionally, the roles and regulation of Rho GTPase signalling networks during cancer cell extravasation will be discussed, both from a cancer cell and endothelial cell point of view.

Keywords: Rho GTPase; cancer; endothelium; extravasation; invadopodia; metastasis.

Figure 1

Schematic overview of metastasis formation to distant organs. (A) Metastasis formation is a complex process consisting of multiple steps: dissociation from the primary tumour (1), local invasion (2), intravasation (3), extravasation (4), formation of a micrometastasis (5) and metastatic colonization (6). Given its complexity, only a small fraction of disseminated tumour cells may ultimately form a metastatic lesion. (B) Extravasation involves many interactions between endothelial cells (ECs) and invasive circulating tumour cells (CTCs). These interactions are mediated by cell-adhesion molecules such as selectins and integrins expressed on their cell surfaces (step 1–3 rolling, adhesion and docking), which may ultimately result in the transmigration of CTCs into the underlying tissue (step 4 transmigration).

Figure 2

Adhesion of leukocytes (upper part) and circulating tumour cells (lower part) to ECs during extravasation. (A) Step 1: rolling/primary attachment. This step is mediated by selectins. Leukocytes express PSGL-1 and L-selectin to directly roll over E- and P-selectin expressing ECs. CTCs, on the other hand, generally do not express these molecules and instead express a wide variety of CAMs to bind to selectins on ECs, or use P-selectin expressing platelets as intermediates. (B) Step 2: firm adhesion. Leukocytes express β1 or β2 integrins (LFA-1, Mac1, VLA-4) to directly firmly adhere to ECs. Some CTCs do express VCAM-1, ICAM-1 and L1-CAM ligands to directly firmly adhere to ECs, but CTCs can also use circulating leukocytes as intermediates by expressing ICAM-1 themselves. CAMs expressed by CTCs are shown in green, CAMs expressed by circulating leukocytes/platelets are shown in red, and CAMs expressed by ECs are shown in black.

Figure 3

The regulation of typical Rho GTPases. Rho GTPases are active at cellular membranes where they function as signal transducers. A guanine nucleotide exchange factor (GEF) exchanges GDP for GTP, thereby bringing Rho in its GTP-bound, active state. A GTPase-activating protein (GAP) hydrolyses Rho GTP, thereby cleaving off a phosphate group and bringing Rho in its GDP-bound, inactive state. A guanine nucleotide dissociation inhibitor (GDI) extracts RhoGDP from the membrane into the cytosol (sequestration), thereby preventing Rho GTPases to be active at cellular membranes. Post-transcriptional and post-translational modifications of Rho GTPases include phosphorylation, ubiquitinylation and miRNA silencing.

Figure 4

The role of Rho GTPases in actin polymerization and cancer cell (transendothelial) migration. Actin polymerization in cellular protrusions mediates migration and extravasation of cancer cells. Cdc42 and Rac1 activate nucleation-promoting factors (NPF) and thus stimulate the formation of a branched actin network via Arp2/3. Cdc42, Rac and Rho activate certain members of the diaphanous-related formin (DRF) family and thus stimulate the formation of a linear actin network. Both Rho-ROCK and Rac/Cdc42-PAK inhibit the activity of cofilin via LIMK, thereby inhibiting actin depolymerization.

Figure 5

The endothelial barrier. Tight junctions (TJs), adherens junctions (AJs) and additional adhesion molecules such as PECAM-1 form the endothelial barrier. TJs mainly consist of occludins, claudins and junctional adhesion molecules (JAMs). AJs mainly consist of VE-cadherin. Adhesion molecules in TJs or AJs are connected to the actin cytoskeleton in ECs via linker proteins, thereby forming a tight barrier that is supported by a basement membrane.

Figure 6

How cancer cells cross the endothelial barrier and the role of Rho GTPases. Cancer cells induce intracellular signalling in ECs upon adhesion and by secreting various factors, which together contribute to local disruption of the endothelial barrier. Additionally, cancer cells recruit circulating leukocytes and/or platelets, which also contribute to extravasation of cancer cells. Tyrosine and serine phosphorylation of VE-cadherin (left) by Src, FAK and/or PAK can be induced via the secretion of IL-8 and VEGF, combined with integrin-mediated adhesion of cancer cells. Stress fibre formation and actomyosin contractility are centrally regulated by Src and RhoA-ROCK signalling (right). Finally, necroptosis can be induced in ECs upon APP-DR6 interactions (right).